Glycolysis inhibition affects proliferation and cytotoxicity of Vγ9Vδ2 T cells expanded for adoptive cell therapy.

BACKGROUND AIMS: Vγ9Vδ2 T cells are under investigation as alternative effector cells for adoptive cell therapy (ACT) in cancer. Despite promising in vitro results, anti-tumor efficacies in early clinical studies have been lower than expected, which could be ascribed to the complex interplay of tumor and immune cell metabolism competing for the same nutrients in the tumor microenvironment. METHODS: To contribute to the scarce knowledge regarding gamma delta T-cell metabolism, we investigated the metabolic phenotype of 25-day-expanded Vγ9Vδ2 T cells and how it is intertwined with functionality. RESULTS: We found that Vγ9Vδ2 T cells displayed a quiescent metabolism, utilizing both glycolysis and oxidative phosphorylation (OXPHOS) for energy production, as measured in Seahorse assays. Upon T-cell receptor activation, both pathways were upregulated, and inhibition with metabolic inhibitors showed that Vγ9Vδ2 T cells were dependent on glycolysis and the pentose phosphate pathway for proliferation. The dependency on glucose for proliferation was confirmed in glucose-free conditions. Cytotoxicity against malignant melanoma was reduced by glycolysis inhibition but not OXPHOS inhibition. CONCLUSIONS: These findings lay the groundwork for further studies on manipulation of Vγ9Vδ2 T-cell metabolism for improved ACT outcome.

High Intensity Aerobic exercise training and Immune cell Mobilization in patients with lung cancer (HI AIM)-a randomized controlled trial.

BACKGROUND: The increasing role of exercise training in cancer care is built on evidence that exercise can reduce side effects of treatment, improve physical functioning and quality of life. We and others have shown in mouse tumor models, that exercise leads to an adrenalin-mediated increased influx of T and NK cells into the tumor, altering the tumor microenvironment (TME) and leading to reduced tumor growth. These data suggest that exercise could improve immune responses against cancer cells by increase immune cell infiltration to the tumor and potentially having an impact on disease progression. Additionally, there are data to suggest that infiltration of T and NK cells into the TME is correlates with response to immune checkpoint inhibitors in patients. We have therefore initiated the clinical trial HI AIM, to investigate if high intensity exercise can mobilize and increase infiltration of immune cells in the TME in patients with lung cancer. METHODS: HI AIM (NCT04263467) is a randomized controlled trial (70 patients, 1:1) for patients with non-small cell lung cancer. Patients in the treatment arm, receive an exercise-intervention consisting of supervised and group-based exercise training, comprising primarily intermediate to high intensity interval training three times per week over 6 weeks. All patients will also receive standard oncological treatments; checkpoint inhibitors, checkpoint inhibitors combined with chemotherapy or oncological surveillance. Blood samples and biopsies (ultrasound guided), harvested before, during and after the 6-week training program, will form basis for immunological measurements of an array of immune cells and markers. Primary outcome is circulating NK cells. Secondary outcome is other circulating immune cells, infiltration of immune cells in tumor, inflammatory markers, aerobic capacity measured by VO2 max test, physical activity levels and quality of life measured by questionnaires, and clinical outcomes. DISCUSSION: To our knowledge, HI AIM is the first project to combine supervised and monitored exercise in patients with lung cancer, with rigorous analyses of immune and cancer cell markers over the course of the trial. Data from the trial can potentially support exercise as a tool to mobilize cells of the immune system, which in turn could potentiate the effect of immunotherapy. TRIAL REGISTRATION: The study was prospectively registered at ClinicalTrials.gov on February 10th 2020, ID: NCT04263467. https://clinicaltrials.gov/ct2/show/NCT04263467.

Vγ9Vδ2 T Cells Concurrently Kill Cancer Cells and Cross-Present Tumor Antigens.

The human Vγ9Vδ2 T cell is a unique cell type that holds great potential in immunotherapy of cancer. In particular, the therapeutic potential of this cell type in adoptive cell therapy (ACT) has gained interest. In this regard optimization of in vitro expansion methods and functional characterization is desirable. We show that Vγ9Vδ2 T cells, expanded in vitro with zoledronic acid (Zometa or ZOL) and Interleukin-2 (IL-2), are efficient cancer cell killers with a trend towards increased killing efficacy after prolonged expansion time. Thus, Vγ9Vδ2 T cells expanded for 25 days in vitro killed prostate cancer cells more efficiently than Vγ9Vδ2 T cells expanded for 9 days. These data are supported by phenotype characteristics, showing increased expression of CD56 and NKG2D over time, reaching above 90% positive cells after 25 days of expansion. At the early stage of expansion, we demonstrate that Vγ9Vδ2 T cells are capable of cross-presenting tumor antigens. In this regard, our data show that Vγ9Vδ2 T cells can take up tumor-associated antigens (TAA) gp100, MART-1 and MAGE-A3 - either as long peptide or recombinant protein - and then present TAA-derived peptides on the cell surface in the context of HLA class I molecules, demonstrated by their recognition as targets by peptide-specific CD8 T cells. Importantly, we show that cross-presentation is impaired by the proteasome inhibitor lactacystin. In conclusion, our data indicate that Vγ9Vδ2 T cells are broadly tumor-specific killers with the additional ability to cross-present MHC class I-restricted peptides, thereby inducing or supporting tumor-specific αßTCR CD8 T cell responses. The dual functionality is dynamic during in vitro expansion, yet, both functions are of interest to explore in ACT for cancer therapy.

The capacity of CD4+ Vγ9Vδ2 T cells to kill cancer cells correlates with co-expression of CD56.

Human Vγ9Vδ2 T cells are a unique T-cell type, and data from recent studies of Vγ9Vδ2 T cells emphasize their potential relevance to cancer immunotherapy. Vγ9Vδ2 T cells exhibit dual properties since they are both antigen-presenting cells and cytotoxic toward cancer cells. The majority of Vγ9Vδ2 T cells are double-negative for the co-receptors CD4 and CD8, and only 20-30% express CD8. Even though they are mostly neglected, a small fraction of Vγ9Vδ2 T cells also express the co-receptor CD4. Here the authors show that CD4+ Vγ9Vδ2 T cells comprise 0.1-7% of peripheral blood Vγ9Vδ2 T cells. These cells can be expanded in vitro using zoledronic acid, pamidronic acid or CD3 antibodies combined with IL-2 and feeder cells. Unlike most conventional CD4+ αß T cells, CD4+ Vγ9Vδ2 T cells are potently cytotoxic and can kill cancer cells, which is here shown by the killing of cancer cell lines of different histological origins, including breast cancer, prostate cancer and melanoma cell lines, upon treatment with zoledronic acid. Notably, the killing capacity of CD4+ Vγ9Vδ2 T cells correlates with co-expression of CD56.

Adrenergic Signaling in Immunotherapy of Cancer: Friend or Foe?

The incidence of cancer is increasing worldwide, which is to a large extent related to the population's increasing lifespan. However, lifestyle changes in the Western world are causative as well. Exercise is intrinsically associated with what one could call a "healthy life", and physical activity is associated with a lower risk of various types of cancer. Mouse models of exercise have shown therapeutic efficacy across numerous cancer models, at least in part due to the secretion of adrenaline, which mobilizes cells of the immune system, i.e., cytotoxic T and natural killer (NK) cells, through signaling of the ß-2 adrenergic receptor (ß2AR). Clinical trials aiming to investigate the clinical value of exercise are ongoing. Strikingly, however, the use of ß-blockers-antagonists of the very same signaling pathway-also shows signs of clinical potential in cancer therapy. Cancer cells also express ß-adrenergic receptors (ßARs) and signaling of the receptor is oncogenic. Moreover, there are data to suggest that ß2AR signaling in T cells renders the cell functionally suppressed. In this paper, we discuss these seemingly opposing mechanisms of cancer therapy-exercise, which leads to increased ß2AR signaling, and ß-blocker treatment, which antagonizes that same signaling-and suggest potential mechanisms and possibilities for their combination.

Expansion With IL-15 Increases Cytotoxicity of Vγ9Vδ2 T Cells and Is Associated With Higher Levels of Cytotoxic Molecules and T-bet.

Cancer immunotherapy has shown great advances during recent years, but it has yet to reach its full potential in all cancer types. Adoptive cell therapy (ACT) is now an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle. In this study, we aimed to explore the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro. We could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1,000 U/ml interleukin (IL)-2 and (b) 100 U/ml IL-2 + 100 U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in natural killer (NK) cell marker or activation marker expression, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells were characterized by an increased expression of perforin, granzyme B, and granulysin compared to IL-2-expanded cells. These cytotoxic molecules were not only increased in a resting state, but also released to a greater extent upon target recognition. In contrast, CD107a and cytokine expression did not differ between expansion conditions. However, IL-2/IL-15-expanded Vγ9Vδ2 T cells showed higher levels of transcription factor T-bet expression, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity. These results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

Exercise Oncology and Immuno-Oncology; A (Future) Dynamic Duo.

Recent advances in clinical oncology is based on exploiting the capacity of the immune system to combat cancer: immuno-oncology. Thus, immunotherapy of cancer is now used to treat a variety of malignant diseases. A striking feature is that even patients with late-stage disease may experience curative responses. However, most patients still succumb to disease, and do not benefit from treatment. Exercise has gained attention in clinical oncology and has been used for many years to improve quality of life, as well as to counteract chemotherapy-related complications. However, more recently, exercise has garnered interest, largely due to data from animal studies suggesting a striking therapeutic effect in preclinical cancer models; an effect largely mediated by the immune system. In humans, physical activity is associated with a lower risk for a variety of malignancies, and some data suggest a positive clinical effect for cancer patients. Exercise leads to mobilization of cells of the immune system, resulting in redistribution to different body compartments, and in preclinical models, exercise has been shown to lead to immunological changes in the tumor microenvironment. This suggests that exercise and immunotherapy could have a synergistic effect if combined.

Chemokine receptor engineering of T cells with CXCR2 improves homing towards subcutaneous human melanomas in xenograft mouse model.

Adoptive cell therapy (ACT) using in vitro expanded tumor infiltrating T lymphocytes (TILs) from biopsy material represents a highly promising treatment of disseminated cancer. A crucial prerequisite for successful ACT is sufficient recruitment of transferred lymphocytes to the tumor site; however, despite infusion of billions of lymphocytes, T cell infiltration into the tumor post ACT is limited. By PCR and Luminex analyses we found that a majority of malignant melanoma (MM) cell lines expressed chemokines CXCL1/Groα, CXCL8/IL-8, CXCL12/SDF-1 and CCL2. Concerning expression of the corresponding receptors on T cells, only the IL-8 receptor, CXCR2, was not expressed on T cells. CXCR2 was therefore expressed in T cells by lentiviral transduction, and shown to lead to ligand specific transwell migration of engineered T cells, as well as increased migration towards MM conditioned medium. In vivo homing was assessed in a xenograft NOG mouse model. Mice with subcutaneous human melanoma were treated with MAGE-A3 specific T cells transduced with either CXCR2 or MOCK. Transducing T cells carrying the MAGE-A3a3a high affinity T cell receptor with CXCR2 increased tumor infiltration. Flow cytometry analysis 7 days after ACT showed a doubling in CD3+ T cells in tumor digest of mice receiving CXCR2 transduced T cells compared to MOCK treated mice, a finding confirmed by immunohistochemistry. In conclusion, our CXCR2 transduced T cells are functional in vitro and transduction with CXCR2 increases in vivo homing of T cells to tumor site.